N-Formyl-Methionyl-Leucyl-Phenylalanine Plays a Neuroprotective and Anticonvulsant Role in Status Epilepticus Model.

Status epilepticus (SE) is described as continuous and self-sustaining seizures, which triggers hippocampal neurodegeneration, inflammation, and gliosis. N-formyl peptide receptor (FPR) has been associated with inflammatory process. N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide plays an anti-inflammatory role, mediated by the activation of G-protein-coupled FPR. Here, we evaluated the influence of fMLP peptides on the behavior of limbic seizures, memory consolidation, and hippocampal neurodegeneration process. Male Wistar rats (Rattus norvegicus) received microinjections of pilocarpine in hippocampus (H-PILO, 1.2 mg/µL, 1 µL) followed by fMLP (1 mg/mL, 1 µL) or vehicle (VEH, saline 0.9%, 1 µL). During the 90 min of SE, epileptic seizures were analyzed according to the Racine's Scale. After 24 h of SE, memory impairment was assessed by the inhibitory avoidance test and the neurodegeneration process was evaluated in hippocampal areas. There was no change in latency and number of wet dog shake (WDS) after administration of fMLP. However, our results showed that the intrahippocampal infusion of fMLP reduced the severity of seizures, as well as the number of limbic seizures. In addition, fMLP infusion protected memory dysfunction followed by SE. Finally, the intrahippocampal administration of fMLP attenuated the process of neurodegeneration in both hippocampi. Taken together, our data suggest a new insight into the functional role of fMLP peptides, with important implications for their potential use as a therapeutic agent for the treatment of brain disorders, such as epilepsy. Schematic drawing on the neuroprotective and anticonvulsant role of fMLP during status epilepticus. Initially, a cannula was implanted in hippocampus and pilocarpine/saline was administered into the hippocampus followed by fMLP/saline (A-C). fMLP reduced seizure severity and neuronal death in the hippocampus, as well as protecting against memory deficit (D).

Novel nano-carriers with N-formylmethionyl-leucyl-phenylalanine-modified liposomes improve effects of C16-angiopoietin 1 in acute animal model of multiple sclerosis.

Gradual loss of neuronal structure and function due to impaired blood-brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Bis(4-glycosyloxybenzyl) 2-isobutyltartrate and dihydrophenanthrene derivatives from the pseudobulbs of Pholidota chinensis and their anti-inflammatory activity.

Six previously undescribed components, bis(4-glycosyloxybenzyl) 2-isobutyltartrate derivatives (pholidotoside A-E) and phenolic glycoside (pholidotosin A), together with twenty known compounds were isolated from the pseudobulbs of Pholidota chinensis. Their structures and absolute configuration were elucidated and established through various spectroscopic and chemical methods. The anti-inflammatory potential of selected compounds was examined using a human neutrophil cell model activated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB). Among these, dihydrophenanthrenes exhibited potent inhibitory effect on both superoxide anion generation and elastase release assays with IC50 values ranging from 0.41 ± 0.05 to 7.14 ± 0.30 µM.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples.

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples. Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions. Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively. Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions.

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-ß/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and ß-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

Method for simultaneous tracking of thousands of unlabeled cells within a transparent 3D matrix.

Three-dimensional tracking of cells is one of the most powerful methods to investigate multicellular phenomena, such as ontogenesis, tumor formation or wound healing. However, 3D tracking in a biological environment usually requires fluorescent labeling of the cells and elaborate equipment, such as automated light sheet or confocal microscopy. Here we present a simple method for 3D tracking large numbers of unlabeled cells in a collagen matrix. Using a small lensless imaging setup, consisting of an LED and a photo sensor only, we were able to simultaneously track ~3000 human neutrophil granulocytes in a collagen droplet within an unusually large field of view (>50 mm2) at a time resolution of 4 seconds and a spatial resolution of ~1.5 µm in xy- and ~30 µm in z-direction. The setup, which is small enough to fit into any conventional incubator, was used to investigate chemotaxis towards interleukin-8 (IL-8 or CXCL8) and N-formylmethionyl-leucyl-phenylalanine (fMLP). The influence of varying stiffness and pore size of the embedding collagen matrix could also be quantified. Furthermore, we demonstrate our setup to be capable of telling apart healthy neutrophils from those where a condition of inflammation was (I) induced by exposure to lipopolysaccharide (LPS) and (II) caused by a pre-existing asthma condition. Over the course of our experiments we have tracked more than 420.000 cells. The large cell numbers increase statistical relevance to not only quantify cellular behavior in research, but to make it suitable for future diagnostic applications, too.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the ßγ subunit of Gi-protein of fMLP receptor in human neutrophils.

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein ßγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 µM)-induced superoxide anion production (IC50:1.98 ± 0.14 µM), the release of cathepsin G (IC50:2.76 ± 0.15 µM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the ßγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCß by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the ßγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

Optimization of the Transwell® assay for the analysis of neutrophil chemotaxis using flow cytometry to refine the clinical investigation of immunodeficient patients.

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women.

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.

[Mitoxantrone Inhibits FMLP-Induced Degenerative Changes in Human Neutrophils].

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.

Cherbonolides M and N from a Formosan Soft Coral Sarcophyton cherbonnieri.

Two new isosarcophine derivatives, cherbonolides M (1) and N (2), were further isolated from a Formosan soft coral Sarcophyton cherbonnieri. The planar structure and relative configuration of both compounds were established by the detailed analysis of the IR, MS, and 1D and 2D NMR data. Further, the absolute configuration of both compounds was determined by the comparison of CD spectra with that of isosarcophine (3). Notably, cherbonolide N (2) possesses the unique cembranoidal scaffold of tetrahydrooxepane with the 12,17-ether linkage fusing with a γ-lactone. In addition, the assay for cytotoxicity of both new compounds revealed that they showed to be noncytotoxic toward the proliferation of A549, DLD-1, and HuCCT-1 cell lines. Moreover, the anti-inflammatory activities of both metabolites were carried out by measuring the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion and elastase release in the primary human neutrophils. Cherbonolide N (2) was found to reduce the generation of superoxide anion (20.6 ± 6.8%) and the elastase release (30.1 ± 3.3%) in the fMLF/CB-induced human neutrophils at a concentration of 30 µM.

Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

N-Formyl Methionine Peptide-Mediated Neutrophil Activation in Systemic Sclerosis.

Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc.

A new dimeric protoberberine alkaloid and other compounds from the tubers of Tinospora dentata.

A new dimeric quaternary protoberberine alkaloid, bispalmatrubine (1), and thirteen known compounds (2-14) were purified from the tubers of Tinospora dentata. Their structures were determined by spectroscopic and spectrometric analytical methods. Among the isolates, eight compounds were examined for their in vitro anti-inflammatory potential and several tested alkaloids displayed moderate inhibitory effects of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

Silver nanoparticles and silver ions cause inflammatory response through induction of cell necrosis and the release of mitochondria in vivo and in vitro.

Owing to the excellent antibacterial and antiviral activity, silver nanoparticles have a widespread use in the food and pharmaceutical industries. With the increase in the production and use of the related products, the potential hazard of silver nanoparticles has aroused public attention. The main purpose of this study is to explore the toxicity of silver nanoparticles and induction of lung inflammation in vitro and in vivo. Here, we validated that small amounts of silver ions dissolved from silver nanoparticles caused the depolarization of plasma membrane, resulting in an overload of intracellular sodium and calcium, and eventually led to the cell necrosis. The blockers of calcium or sodium channels inversed the toxicity of silver ions. Then, we instilled silver nanoparticles or silver nitrate (50 µg per mouse) into the lungs of mice, and this induced pulmonary injury and mitochondrial content release, led to the recruitment of neutrophils to the lung tissue via p38 MAPK pathway. Altogether, these data show that released silver ions from nanoparticles induced cell necrosis through Na+ and Ca2+ influx and triggered pulmonary inflammation through elevating mitochondrial-related contents released from these necrotic cells.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

N-Formylated Peptide Induces Increased Expression of Both Formyl Peptide Receptor 2 (Fpr2) and Toll-Like Receptor 9 (TLR9) in Schwannoma Cells-An In Vitro Model for Early Inflammatory Profiling of Schwann Cells.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCß. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.

Insights into the Activation Mechanism of the ALX/FPR2 Receptor.

The formyl peptide receptor 2 (ALX/FPR2), a G-protein-coupled receptor (GPCR), plays an important role in host defense and inflammation. This receptor can be driven as pro- or anti-inflammatory depending on its agonist, such as N-formyl-Met-Leu-Phe-Lys (fMLFK) and resolvin D1 (RvD1) or its aspirin-triggered 17 (R)-epimer, AT-RvD1, respectively. However, the activation mechanism of ALX/FPR2 by pro- and anti-inflammatory agonists remains unclear. In this work, on the basis of molecular dynamics simulations, we evaluated a model of the ALX/FPR2 receptor activation process using two agonists, fMLFK and AT-RvD1, with opposite effects. The simulations by both fMLFK and AT-RvD1 induced the ALX/FPR2 activation through a set of receptor-core residues, in particular, R205, Q258, and W254. In addition, the activation was dependent on the disruption of electrostatic interactions in the cytoplasmic region of the receptor. We also found that in the AT-RvD1 simulations, the position of the H8 helix was similar to that of the same helix in other class-A GPCRs coupled to arrestin. Thus our results shed light on the mechanism of activation of the ALX/FPR2 receptor by pro-inflammatory and pro-resolution agonists.